Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK−/− fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK−/− cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK−/− v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK–Src-p130Cas–Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways.

Localized chromatin modifications of histone tails play an important role in regulating gene transcription, and aberration of these processes leads to carcinogenesis. Methylated histone lysine residues, a key player in chromatin remodeling, are demethylated by the JmjC class of enzymes. Here we show that JMJD5 (now renamed KDM8), a JmjC family member, demethylates H3K36me2 and is required for cell cycle progression. Chromatin immunoprecipitation assays applied to human genome tiling arrays in conjunction with RNA microarray revealed that KDM8 occupies the coding region of cyclin A1 and directly regulates transcription. Mechanistic analyses showed that KDM8 functioned as a transcriptional activator by inhibiting HDAC recruitment via demethylation of H3K36me2, an epigenetic repressive mark. Tumor array experiments revealed KDM8 is overexpressed in several types of cancer. In addition, loss-of-function studies in MCF7 cells leads to cell cycle arrest. These studies identified KDM8 as an important cell cycle regulator.egulation of gene expression through posttranslational modification of the core histones has increasingly shown to be of great importance, particularly in a cancer setting. Among the multiple types of histone modifications, histone methylation, once considered irreversible, has quickly emerged to become a key epigenetic mark in regulating many critical cellular functions. The recent discovery of histone demethylases has shed light on the reversibility of this chromatin mark and its effects on gene expression. Studies exploring the JmjC (Jumonji C domain)-containing proteins, a newclass of histone demethylases (1-4), primarily identified their enzymatic activity at the promoters of specific target genes (5, 6).The JmjC domain-containing gene family encodes a wide range of the eukaryotic genome and is conserved in species spanning from yeast to humans. Currently, most family members classified as histone demethylases contain known histone-binding domains such as PHD and Tudor domains (7). JMJD5 (renamed KDM8) is a member of this extensive protein family that lacks recognizable histone-binding domains and remains largely unexplored. Although one study speculated that KDM8 acts as a potential tumor suppressor gene based on retrovirus insertional mutagenesis (8), no biological and molecular characterizations were described in the report.We extensively examine and provide evidence that KDM8 possesses H3K36me2 demethylase activity and has the ability to regulate cyclin A1 transcription in MCF7 breast cancer cells. We found that KDM8 is recruited to cyclin A1 coding region bound H3K36me2 and demethylates this mark, resulting in increased transcriptional activity. This finding is a departure from previous studies that showed that the majority of histone demethylases exert their epigenetic effects at the promoters of genes. Additionally, we describe overexpression of KDM8 in breast cancer tumors as well as its requirement for MCF7 cell cycle progression. ResultsJMJD5/KDM8 Is a H3K36me2 Demethylase. Our initial e...

Focal adhesion kinase (FAK) was first identified as a viral Src (v-Src) substrate, but the role of FAK in Src transformation events remains undefined. We show that stable expression of the FAK C-terminal domain (termed FRNK) in v-Src-transformed NIH 3T3 fibroblasts inhibited cell invasion through Matrigel and blocked experimental metastases in nude mice without effects on cell motility. FRNK inhibitory activity was dependent upon its focal contact localization. FRNK expression disrupted the formation of a v-Src-FAK signaling complex, inhibited p130Cas tyrosine phosphorylation, and attenuated v-Src-stimulated ERK and JNK kinase activation. However, FRNK did not affect v-Src-stimulated Akt activation, cell growth in soft agar, or subcutaneous tumor formation in nude mice. FRNK-expressing cells exhibited decreased matrix metalloproteinase-2 (MMP-2) mRNA levels and MMP-2 secretion. Transient FRNK expression in human 293 cells inhibited exogenous MMP-2 promoter activity and overexpression of wild-type but not catalytically-inactive (Ala-404) MMP-2 rescued v-Src-stimulated Matrigel invasion in the presence of FRNK. Our findings show the importance of FAK in Src-stimulated cell invasion and support a role for Src-FAK signaling associated with elevated tumor cell metastases.

SummaryCell migration plays an important role in embryonic development, wound healing, immune responses, and in pathological phenomena such as tissue invasion and metastasis formation. In this review, we summarize recent reports that connect the focal adhesion kinase (FAK) to cell migration and invasion.

Several vascular diseases result from neointima formation, a process that is characterized by the accumulation of vascular smooth muscle cells (SMCs) 1 and extracellular matrix (ECM) proteins in the intima of blood vessels (1). Neointima formation is triggered upon damage to the endothelial lining by the local release of chemotactic cytokines or growth factors (2) and by increased production of ECM proteins (3). Because combinations of these factors can stimulate cell division, it was originally hypothesized that neointimal hyperplasia resulted from enhanced SMC proliferation (4). However, further studies have shown that SMC migration also plays a significant role in neointima formation (5). Growth factors acting as chemotactic agents (6) and ECM molecules acting as haptotactic factors (7) can independently stimulate SMC migration. Therefore, a common signaling component that coordinates both chemotactic and haptotactic cell migration events would be a promising target for intervention strategies.Investigations of the molecular regulation of cell migration have demonstrated an important role for the focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK) that localizes to cell-substratum contact sites also known as focal adhesions (for a review see Ref. 8). Genetic support for FAK in promoting cell motility comes from studies using FAK-null fibroblasts that exhibit refractory responses to normal motility-promoting stimuli (9). Importantly, these defects are rescued by stable re-expression of FAK in FAK-null cells (10, 11). Work from a number of laboratories using a variety of cell types has provided evidence that FAK promotes cell migration potentially through the activation of multiple downstream targets such as Src family PTKs (10,14), by the increased phosphorylation of p130 Cas (15,16) or paxillin adaptor proteins (17), or through the association with other signaling proteins such as Grb7 (18) and . Although no clear consensus has emerged on the molecular mechanism(s) of how FAK promotes cell migration, studies with FAK-null cells have shown that FAK is required for both integrin-and growth factor-stimulated cell motility (10, 12).FAK activity is regulated by protein-tyrosine phosphatase action (16) and inhibited by the overexpression of the FAK C-terminal domain termed FRNK (FAK-related non-kinase) (22)(23)(24). Importantly, the regulated and autonomous expression of FRNK during embryonic development is under the control of alternative intronic promoter region and therefore may function as an endogenous inhibitor of FAK in vivo (22,25) In this study, we employed the transient and stable overexpression of hemagglutinin (HA)-tagged FRNK to investigate the role of endogenous FAK in promoting platelet-derived